Novel pulp capping material based on sodium trimetaphosphate: synthesis, characterization, and antimicrobial properties

Abstract Objectives: To evaluate the mechanical, physicochemical, and antimicrobial properties of four different formulations containing micro- or nanoparticles of sodium trimetaphosphate (mTMP and nTMP, respectively). Methodology: Four experimental groups were used in this investigation: two mTMP groups and two nTMP groups, each containing zirconium oxide (ZrO2), and solution containing either chitosan or titanium oxide (TiO2) nanoparticles (NPs). Setting time, compression resistance, and radiopacity were estimated. The agar diffusion test was used to assess the antimicrobial activity of the formulations against five different microbial strains: Streptococcus mutans, Lactobacillus casei, Actinomyces israelii, Candida albicans, and Enterococcus faecalis. Parametric and nonparametric tests were performed after evaluating homoscedasticity data (p<0.05). Results: From the properties evaluated, nTMP cements required less setting time and showed greater resistance to compression. Cements containing TiO2 showed greater radiopacity for both nTMP and mTMP. All four cement formulations showed antimicrobial activity against S. mutans and L. casei Conclusion: Formulations containing nTMP have shorter setting times and higher compressive strength, and those with TiO2 nanoparticles showed antimicrobial activities. Clinical relevance: The cement containing nTMP, ZrO2, and TiO2 could be an alternative material for protecting the pulp complex.


Introduction
The most common causes of injuries to pulp tissue are deep cavities due to dental caries and dental trauma.
If injured, the maintenance of pulp tissue integrity is achieved by pulp therapy. It aims to maintain, even if only partially, pulp vitality by eliminating bacteria from the dentin-pulp complex 1 and preserve its functional and biological activities. Frequently used therapies are indirect and direct pulp capping. In direct pulp capping, the dental pulp is exposed, and a protective agent that induces the repair of hard tissue is used. In indirect pulp capping, as the pulp remains unexposed, a thin layer of material is applied on the dentin. 2 To protect the pulp complex and preserve its vitality, an ideal pump capping material should be able to provide an optimal seal, minimize microleakage, and show low solubility, excellent bioactivity, dimensional stability, bactericidal properties, radiopacity, and high compression resistance. 3 Different materials have been used in direct pulp capping, such as calcium hydroxide (Ca(OH) 2 ) paste and mineral trioxide aggregate (MTA), because they offer excellent antimicrobial properties (pH ~ 12) and can promote the formation of a mineralized tissue barrier. [4][5][6] Clinically, Ca(OH) 2 paste is easy to handle and has an optimal setting time, but its high solubility produces a poor seal and it lacks adhesive properties. 7,8 In contrast, MTA shows low solubility and excellent marginal sealing, but a lower antimicrobial activity than Ca(OH) 2 . 9,10 Moreover, MTA has significant clinical disadvantages, such as long setting time, [11][12][13] difficult handling, and high cost. 1, [14][15][16] Currently, there are various MTA-based products available on the market, with setting times ranging from minutes to hours, and some of them offer easy handling.
Sodium cyclophosphates, such as sodium trimetaphosphate (TMP), can preserve the stability and integrity of the enamel mineral surface, 17 nucleate calcium ions, 18 increase enamel remineralization, 18 and obliterate dentinal tubules if associated with fluoride. 19 Moreover, studies have shown that a reduction in TMP particle size increases their anticaries potential. [20][21][22][23][24] In view of the need for materials that show better mechanical, physicochemical, and antimicrobial properties, this study aimed to develop a cement containing micro-(mTMP) or nanoparticulate sodium trimetaphosphate (nTMP) and evaluate the effects of trimetaphosphate microparticles (mTMP) and nanoparticles (nTMP) on the physicomechanical, physicochemical, and antimicrobial properties of its four different formulations.

Cement with micro-or nanoparticle TMP
Our novel cement consists of a powder containing mTMP or nTMP 20 , zirconium oxide (ZrO 2 ) as a radiopacifier, an aqueous solution containing an emulsifier, and either titanium oxide (TiO 2 ) or chitosan NPs. In the development of this material, depending on the proportion of the different components, the material either failed to set and/or expanded or contracted during hardening. Thus, at this stage of development, setting times and dimensional changes were considered. Four different formulations were prepared: (1) mTMP, ZrO 2 , and chitosan NPs (ZMC); (2) nTMP, ZrO 2 , and chitosan NPs (ZNC); (3) mTMP, ZrO 2 , and TiO 2 NPs (ZMT); and (4) nTMP, ZrO 2 , and TiO 2 NPs (ZNT). Percentages for each constituent of the powder formulations and the powder/liquid ratio (P/L) are shown in Table 1. As the consistency of a dental cement can vary according to its application, the powder/liquid ratio indicated in Table 1  ADA specification no. 57. In total, five samples were prepared for each cement formulation using a stainless-steel ring of 10 mm internal diameter and 2 mm thickness. The assembly, comprising the mold and test material, remained during the specified setting time in a cabinet at 37°C and with a relative humidity between 95 and 100%. Three minutes after the start of mixing, an indenter needle (Gillmore) with a mass of 100.0±0.5 g and a flat tip of 2.0±0.1 mm in diameter, was carefully lowered vertically onto the surface of the sample. This procedure was repeated every 30 seconds until the needle failed to make a complete circular indentation on the test material.
The time period that elapsed from start of mixing to when the needle failed to make a complete circular indentation on the tested material surface determined the setting time.

Compressive strength
For each cement formulation, 10 specimens were prepared using a cylindrical mold 4 mm in diameter and 6 mm in height. Each cement specimen was mixed on a glass plate with a steel spatula, and the obtained paste was inserted into the mold, which was supported under a microscope slide. Next, the assembly was stored for seven days in a cabinet at 37±1ºC and a relative humidity between 95 and 100%. After this storage period, the samples were removed from the molds for compressive strength measurements using a universal testing machine

Radiopacity
Radiopacity tests were performed according to ANSI/ADA specification no. 57. For each cement formulation, three samples were prepared using a mold 1 mm thick and 10 mm in diameter. Each sample was positioned in the center of a dental intraoral X-ray sensor adjacent to an aluminum step wedge.
The set was exposed to X-ray radiation emitted by an submitted to one-way variance analysis, followed by a Student-Newman-Keuls test. Data from antimicrobial tests showed a heterogeneous distribution and were submitted to a Kruskal-Wallis test, followed by a Student-Newman-Keuls test. Table 2 shows the values obtained for setting time. Using nTMP, instead of mTMP, in the cement formulation reduced setting times in a little more than 50%. The TiO 2 groups showed a shorter setting time than the chitosan groups. The results obtained in the compressive strength test showed greater resistance to compression in groups containing nTMP with either TiO 2 or chitosan NPs (Table 2). Groups with chitosan NPs showed higher compressive strength values (MPa) than those with TiO 2 , regardless of particle size.

Results
The cement with the greatest compressive strength was ZNC (p<0.05). We found statistically significant differences in the compressive strength of the groups analyzed, whose mean values ranged from 2.24±0.41     As part of the continuous evolution in conservative dentistry, there has been motivation for research to further investigate the possibilities of inducing repair and regeneration of lost hard dental tissue. Phosphates, such as sodium trimetaphosphate, can attract calcium ions, and, therefore, act as nucleating agents for apatite crystals. 28,29 In particular, this process can induce mineralization of dentinal tissue, 30,31 in addition to stimulating dental tissue repair and regeneration.
Ideal physicochemical properties are essential for a dental material to be suitable for clinical use.
These physicochemical attributes will generally dictate the clinical indications of the material. Setting time is an important parameter for dental healthcare professionals since it represents the time interval available for the clinical procedure to be performed.
There is a statistically significant difference in the mean setting time values among treatment groups (p<0.001). Using nanoparticulate TMP in cement formulations led to a reduction of 55. 7 and 53.4% in the setting times of the cements with TiO 2 and chitosan NPs, respectively. It is well known that particle size affects certain physical and chemical properties of cements. 32,33 Specifically, the finer the cement Although studies show that TMP, associated with fluoride, reduces the mineral loss of dental structures (confirmed by a clinical study in children 40 ) and that a reduction in particle size has been shown to increase its anti-caries potential, TMP lacks antimicrobial or antifungal activity. 41  Regarding the other microorganisms evaluated, A. israelii, E. faecalis, and C. albicans, the groups evaluated showed no antimicrobial action. The explanation for the non-inhibition of both bactericidal agents is related to numerous factors arising from the characteristics and structures of the bacteria.
A. israelii, despite being classified as gram-positive, showed resistance to antimicrobial action due to its morphology and resistance to oxygen, which proved that this microorganism shows irregular filaments.
This difference in its filaments increases the structural strength of the bacterium and contributes to its resistance. E. faecalis has proteins on its surface that differ from other bacteria and give it the strength to survive in different environments and against different drugs and bactericidal agents. 43 In regard to C. albicans, non-inhibition may be due to the more complex cell structure of yeasts and hyphae compared to bacteria, which possibly hampers the effects of chitosan and TiO 2 nanoparticles. This investigation indicates that the formulation containing nTMP, ZrO2, and TiO 2 nanoparticles showed the best results due to its lowest setting time, high compressive strength, and antimicrobial activity in relation to S. mutans, which was very close to the ZMT formulation.